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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3889
pigenetic information encompasses DNA methylation, noncoding RNA and modiﬁcations to histones. It serves a
critical function in the regulation of gene expression and
thus development and disease. From one generation to the next,
the epigenome is reprogrammed in the gametes and the embryo
to allow for totipotency and to prevent the transmission of
epigenetic errors. However, not all regions of the epigenome are
reset, permitting the transmission of epigenetic information from
parents to offspring. It has been postulated that epigenetic
information passed on by the gametes allows for the transmission
of information on parental environmental exposures. The
underlying mechanisms implicated in epigenetic inheritance are
unknown, and to date there is a lack of mechanistic evidence to
support epigenomic transmission via the sperm.
DNA methylation is the most studied epigenetic modiﬁcation
and takes place at the 5-position of cytosine residues within CpG
dinucleotides and is essential for the heritable silencing and
regulation of many types of DNA sequences; it occurs at about
30 million sites throughout the mammalian genome1. DNA
methylation is involved in regulating genes that are subject to
parent-of-origin imprinting. These patterns of genomic
methylation are acquired in the germline beginning in utero
and are completed during spermatogenesis and passed to the
offspring. DNA methylation is catalysed by a family of DNA
(cytosine-5)-methyltransferases and cooperates with histone
modiﬁcations in the regulation of gene expression and silencing.
It has long been known that disease can be transmitted via the
genetic layer but new modes of inheritance via the epigenome are
being discovered2. Whereas an organism’s genotype is relatively
static throughout life, the epigenome is highly dynamic and can
adapt, or be altered, in response to the internal or external
environment. Recent studies suggest that there are critical
windows in development where the epigenome is susceptible
to the introduction of epimutations through exposure to
environmental factors such as toxicants or diet3. Very recent
studies strongly suggest that paternal transmission of disease can
occur via the epigenetic layer in sperm. Chronic high-fat diets fed
to Sprague–Dawley rat sires induced glucose intolerance and
impaired insulin secretion in their adult female offspring4. In
humans, nutrition in males just before puberty can alter
descendents survival, and predisposition to diabetes and
cardiovascular disease5. In these prior studies, the sperm
epigenome was not analysed.
Dietary components serve to modulate the availability of
methyl donors for methylation reactions. The B vitamins serve as
coenzymes of one carbon (C1) metabolism, which is a network of
biochemical reactions in which a C1 unit is received from methyl
donor nutrients and used in the methylation of DNA, RNA and
proteins. Thus, agents that modulate C1 metabolism directly or
indirectly affect levels of S-adenosyl methionine, the principal
methyl donor for the methylation of DNA. Thus, dietary folate
levels can directly inﬂuence levels of cellular DNA methylation
and consequently affect gene expression6.
Spermatogenesis consists of a carefully coordinated series of
events beginning with division of spermatogonia, followed by
meiosis to produce haploid spermatids, and ﬁnally the differentiation of the spermatids into mature spermatozoa. During
spermatogenesis, there are epigenetic processes unlike those in
the development of any other cell type, including a massive and
active demethylation of the genome to allow for its sex-speciﬁc
resetting by a combination of DNA methylation and histone
modiﬁcations7. During mammalian gamete formation, some
genes acquire heritable molecular imprints through epigenetic
demarcation that act to either suppress or activate the expression
from one parental allele in the somatic cells of offspring. These
imprints are established in a sex-speciﬁc manner in the forming
2
gametes so that the gene ‘remembers’, so to speak, its parent of
origin in the offspring. If imprinting of genes is disturbed during
gametogenesis, gene expression is compromised in the resulting
offspring8. The resultant hemizygosity imposed by imprinting is
believed to confer genetic vulnerability, and errors in this process
are associated with diseases such as cancer, Prader–Willi
syndrome and behavioural disorders9. Examination of DNA
methylation in sperm reveals that there are unique distributions
in comparison with somatic cells10. Infertility as a chronic disease
frequently coexists with obesity, metabolic syndrome and
cardiovascular disease. Infertile men have been shown to have
an altered sperm epigenome including altered levels of DNA
methylation, imprinted loci defects, lower pregnancy rates and a
high frequency of abnormal embryos11. To date, there have been
a handful of studies, indicating that there can be paternal
transmission of subtle effects such as metabolic state to offspring.
Paternal diets high in fat or low in protein can alter the
metabolism in offspring and pancreatic gene expression4,12.
However, there is a paucity of evidence delineating the
mechanisms underlying epigenetic inheritance from a father to
offspring. In clinical studies, a positive association between folate
and fertility has been observed13 and infertility is linked with a
mutation in a key enzyme in folate metabolism, 5,10methylenetetrahydrofolate reductase (MTHFR)14. Although
folate supplementation is recommended to women before
pregnancy, and food in North America has been folate fortiﬁed
since 1998 (ref. 15), there are certain physiological states that
coincide with low serum folate. Obesity and polymorphisms in
folate metabolic enzymes reduce the availability of folate for
methyl donation16,17.
Here we hypothesize that the availability of folate will alter the
levels of DNA methylation in spermatogenesis with consequences
for the sperm epigenome and pregnancy outcomes. Using an
inbred C57BL/6 mouse model that was exposed to low dietary
folate beginning in utero and throughout life, we show that
paternal diet alters sperm DNA methylation and is associated
with negative reproductive outcomes including birth defects in
offspring. Remarkably, the sites of the sperm epigenome bearing
changes in DNA methylation are associated to genes implicated
in development and chronic disease. This study is the ﬁrst to
show that the folate status of the father, not just the mother, may
be of equal importance in determining reproductive success in
terms of healthy pregnancy outcomes.
Results
Transient effects of folate deﬁciency on meiosis. Male inbred
C57BL/6 mice received throughout life either the control folatesufﬁcient (FS) diet (2 mg folic acid per kg) that contained the
recommended amount of folate for rodents18, or a folate-deﬁcient
(FD) diet (0.3 mg folic acid per kg, 14.3% of the recommended
amount of folate; Fig. 1a). Dietary exposure began in utero when
epigenetic patterning in germ cells begins19. Testis and body
weights and testis histology were examined from pups at postnatal
days (PND) 6, 10, 12, 14 and 18 corresponding to the appearance
of spermatogenic cell types20. Histological examination of testis at
PND 12, when meiotic cells at the leptotene stage ﬁrst appear,
revealed a delay in meiotic onset in FD pups (Fig. 1b,c,d). There
were no apparent effects of diet on Sertoli (Supplementary Fig. S1)
and Leydig cells (Supplementary Fig. S2). Body weight of male
offspring was monitored as a general gauge of health and there was
no reduction of body weight in FD males compared with FS males.
These ﬁndings are consistent with the C57BL/6 mice model, which
was on the same FD diet for 12–14 months21. Histological
examination of adult testes revealed no detectable morphological
differences between FS and FD mice (Fig. 1f,g). No effects of diet
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Folate deﬁciency increases DNA damage in spermatocytes.
Folate deﬁciency has been linked to increased DNA breaks owing
to excessive uracil incorporation22. The most damaging DNA
breaks in terms of genome integrity are DNA double-strand
breaks (DSBs)23. Terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labelling immunostaining was used to detect
cellular DNA fragmentation and to assess apoptosis. No
spermatogenic stage-speciﬁc apoptosis was observed and the
proportion of apoptotic tubules was not increased in FD males,
in comparison with FS males (P40.05 by Student’s t-test;
Supplementary Fig. S3). As a second assessment of DNA
damage, spermatocyte-enriched cell spreads were prepared
from FS and FD mice. Chromosomes were co-immunolabelled
with the synaptonemal complex protein 3 (SYCP3) and the
phosphorylated histone variant, g-H2AX (Supplementary
Fig. S4). The g-H2AX variant is involved in DNA repair
activities and the targeting of DNA DSBs24. In meiotic
prophase I, g-H2AX foci are present as part of the molecular
mechanisms mediating recombination. The number of g-H2AX
peaks in leptotene spermatocytes with 300 foci per cell
and gradually decreases to 120 foci per cell in early pachytene,
and to 48 in late pachytene25. Consistent with previous studies,
the sex body was intensely stained by g-H2AX (Supplementary
Fig. S4a–d) and the number of foci observed was on average
125.5±2.1 in early pachytene and 58.5±1.9 in late pachytene of
FS25. In early and late pachytene spermatocytes from FD mice, gH2AX foci were signiﬁcantly increased suggesting DNA damage.
As meiotic prophase progressed, the difference in the foci number
between FS and FD cells increased from 5.9% in early pachytene
to 12.5% in late pachytene spermatocytes (Supplementary
Fig. S4e). To determine whether DNA damage in spermatocytes
of FD mice was sustained in the sperm or was repaired, a Comet
assay was carried out on sperm from FS and FD mice to measure
DNA single-strand and DSBs26. The Comet assay revealed no
differences in the DNA fragment level as represented by tail
DNA, tail length and tail extent moment (P40.05 by Student’s ttest; Table 1), indicating that DNA damage in FD spermatocytes
was repaired.
Somatic cells are not affected by folate deﬁciency. To determine
whether somatic cells in the testis were sensitive to folate deﬁciency, we examined Sertoli and Leydig cell development at critical points following their proliferation and differentiation27,28. It
is believed that the total number of Sertoli cells determines the
efﬁciency of spermatogenesis29. Thus, we examined the Sertoli
number at PND10 (FS, n ¼ 3; FD, n ¼ 3), since in mice Sertoli
cells proliferate only in fetal/neonatal period and become almost
quiescent at PND12 (ref. 27). Sertoli cells were identiﬁed by
immunoﬂuorescent labelling with anti-MIS (Mullerian-Inhibiting
¨
Substance) and counted in 10 round tubules per animal in FS and
FD (Supplementary Fig. S1a,b). No difference occurred in Sertoli
cell numbers between FS and FD mice (P40.05 by the Mann–
Whitney U-test; Supplementary Fig. S1c).
Leydig cells are prominent in the interstitial space and are
responsible for testosterone production30. In mice, the fetal
Table 1 | Assessment of sperm chromatin integrity for the
folate-sufﬁcient (FS) and deﬁcient (FD) mice by Comet
assay.
Diet
FS
FD
Tail length
99.82±0.44
99.87±0.80
Tail DNA
34.12±0.17
34.06±0.35
Tail extent moment
63.28±0.40
63.35±0.84
Means±s.e.m. of four determinations are shown for each group.
4
Leydig cells are present and observable as round to oval shaped
and are found exclusively in clusters. Leydig cells start to regress
postnatally marked by the scattering of clusters and their
decreasing number28,31. The histological examination of testis
cross-sections stained by the Leydig cell marker, 3b-HSD (3-bHydroxysteroid Dehydrogenase/D-5-4 isomerase), revealed no
detectable size or quantitative differences of fetal Leydig cell
clusters between FS and FD males at PND6 (Supplementary Fig.
S2). Adult Leydig cells (ALD) ﬁrst appear at PND10–13. The
ﬁrst stage of ALDs, from the initiation to PND25, is the
progenitor Leydig cell (PLD) stage, which has a high
proliferation rate. After this stage, ALDs only double once and
then become quiescent31. Thus, we assessed the postnatal ALD
number to determine whether ALD proliferation was affected by
folate deﬁciency. In PND12 FS and FD mice testis, the number
of PLD was quantiﬁed. PLD were identiﬁed as 3b-HSD-positive
cells with slender-shaped cytoplasm and an elongated nucleus,
located singly around seminiferous epithelium (Supplementary
Fig. S2a,b). The ratio of the total number of PLDs to the number
of tubules counted was calculated and compared between FS and
FD mice (FS, n ¼ 3; FD, n ¼ 3; Supplementary Fig. S2c). No
observable change in morphology or number of the PLDs
between FD and FS testis occurred.
Folate deﬁciency leads to negative reproductive outcomes. To
determine whether paternal FD diets affected fertility and
pregnancy outcomes, FS and FD males were mated to reproductively robust outbred CD-1 females that received control diets.
The fertility of FD males was compromised, as demonstrated by
the signiﬁcantly reduced pregnancy rate of 52.38% compared
with 85% for FS mice (Supplementary Fig. S5a). There was no
difference in mating behaviour as evidenced by plug-positive
females but there was an increased breeding interval in females
bred to FD males. We next assessed embryo loss and
development at 18.5 days post conception (dpc). Litters were
sired by FS (n ¼ 32) or FD (n ¼ 35) males. Embryo weight and
crown-rump length were not affected by paternal diet
(Supplementary Table S2). Preimplantation loss was not affected
by diet but post-implantation loss increased in FD-sired
pregnancies (Supplementary Table S3). This increased loss was
reﬂected in the twofold greater resorption rate in pregnancies
sired by FD males in comparison with those sired by FS males
(Supplementary Fig. S5b).
Developmental abnormalities were observed with a greater
frequency in fetuses sired by FD males (Fig. 2; Supplementary
Table S2 and Supplementary Fig. S6). Of FD-sired fetuses, 27%
had visible gross anatomical abnormalities versus 3% in litters
sired by FS fathers (Supplementary Table S2). Malformations
observed in FD-sired offspring included craniofacial abnormalities such as hydrocephalus (Fig. 2b). Limb defects included
underdeveloped digits, or in some cases anonychia and/or hind
limb hyperextension (Fig. 2c). In several FD-sired fetuses,
abnormalities included muscle and/or skeletal defects in the
region of the spine or scapula (Fig. 2d,e). These were conﬁrmed
by histopathological analysis that revealed muscular dysplasia
adjacent to the scapula (Fig. 2f,g). Given that the observed gross
malformations indicated probable skeletal malformations,
selected FS and FD fetuses were processed for skeletal analysis
(Fig. 2h–l). Skeletal analysis conﬁrmed abnormalities such as
reduced ossiﬁcation of the skull as well as a delay in the
development of the digits in FD-sired offspring (Fig. 2h,i). In
addition, misalignment or even incomplete development of the
sternebrae plates was often observed in FD-sired offspring
(Fig. 2j–l). Abnormalities observed in FS-sired offspring were
minor and included a runt and skin discolouration.
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FS
FD
FD
FD
FD
FS
IP
Fr
Pa
FS
FD
Pa
Fr
FD
SO
FS
FD
FD
Figure 2 | Paternal folate deﬁciency increases birth defects in offspring. Fetuses sired by FD males displayed increased developmental abnormalities
indicated by arrows (b–e) than those sired by FS males (a). Shown are the following: (b) hydrocephalus and associated craniofacial defects, (c) limb
hyperextension with dysgenesis of digits, (d) spine malformation and (e) dorsal malformations. Histopathological analysis was carried out on selected FS(f) and FD- (g) sired fetuses. (g) In this thoracic transverse section of an FD-sired fetus, the arrow indicates an imbalance between the right and left side
muscular and bone tissues indicated muscular dysplasia. Scale bars, 1 mm (f,g). Skeletal staining was performed in both FS- (h,j) and FD- (i,k,l) sired
fetuses. Bone is stained purple and cartilage blue. (i) FD-sired fetus lacking interparietal (IP) and supraoccipital (SO) bones and had underdeveloped frontal
(Fr), parietal (Pr) bones and digits. FD-sired fetuses, with misaligned (k), or incomplete development (l) of the sternebrae plates.
Abnormalities in FD pregnancies were also detected at the level
of the placenta. Development of the placenta in part depends on
epigenetic information for genomic imprinting from the father32.
While placenta weight and size did not differ between the FS and
FD offspring (Supplementary Table S2), there were two
occurrences of fused placentas in pregnancies sired by FD
fathers (Supplementary Fig. S6a). The embryos sharing the
placenta were separately connected, but in both cases one embryo
was smaller in size indicating that its development was
compromised by the placental abnormality. Histopathology
conﬁrmed placenta abnormalities at the cellular level where
there was an absence of the giant cell layer and an abnormally
thin spongiotrophoblast layer in a pregnancy sired by a FD male
(Supplementary Fig. S6b,c).
A FD diet alters the sperm epigenome. The sperm epigenome at
the level of DNA methylation was assessed to determine whether
folate availability altered the sperm epigenome, and whether there
were epigenetic alterations at genes implicated in development
and disease that correlated with the abnormal phenotypes
observed in FD-sired offspring examined. Genome-wide DNA
methylation was assessed by methylated DNA immunoprecipitation (MeDIP) followed by hybridization using the NimbleGen
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5mC (log2 ratio MeDIP/input)
mouse 2.1 deluxe promoter array (MeDIP-chip). The sperm cells
were isolated using a swim up method, which prevents contamination by somatic cells. As further validation of the purity of
the sperm population, and to validate the MeDIP procedure, we
assessed enrichment following DNA methylation selection by
MeDIP followed by real-time PCR. Regions selected for analysis
were known to be fully methylated, or not, in spermatozoa
(Supplementary Fig. S7). Gene regions targeted included the
following: ﬁrst, the imprinting control region of H19, the paternally imprinted gene that is hypermethylated in sperm33, second,
the promoter region of Nanog34 also hypermethylated in sperm
and, third, the promoter regions of the testis-speciﬁc variants of
histones H1 (H1t) and H2b (TsH2b), which are hypomethylated
in sperm and hypermethylated in somatic tissues35. We observed,
before and after ampliﬁcation with a WGA2 kit, a strong
enrichment in the MeDIP fraction of H19ICR and Nanog,
whereas H1t and tsH2b displayed very low signals. These results
validate the purity of the sperm population and the speciﬁcity of
the MeDIP before and after the ampliﬁcation process.
By this MeDIP-chip analysis under highly stringent criteria
(false discovery rate (FDR) threshold of 0.1), 57 genomic regions
had altered methylation proﬁles in sperm from FD males in
comparison with sperm from FS (Supplementary Table S4).
Within these regions, methylation differences ranged from
À 1.33 to 2.76 (log2 fold-difference). Forty-six of these regions
were located within 10 kb of the transcription start site and 31
within 5 kb. The regions beyond 10 kb were included in our
analysis, as the existence of a long distance enhancer or silencer
regulated by DNA methylation has been shown to inﬂuence gene
expression as far as 100 kb from the gene36. Indeed,
the methylation level of the differentially methylated region
located in H19 promoter at more than 70 kb from Igf2 regulates
its expression37. Some gene promoter regions had multiple,
dispersed sites of differential methylation such as the promoter of
the microRNA Mir715 (Fig. 3a). Other promoter regions of genes
such as Ddx58 and Sﬁ1 had localized methylation changes close to
the transcription start site (Fig. 3b,c). Interestingly, the
methylation levels were decreased in the FD fraction for certain
genes (Fig. 3a,c) but for others it was increased (Fig. 3b). At
an FDR threshold of 0.25, a further 200 additional differentially
methylated regions were identiﬁed and these regions were
included in functional analysis using gene sets obtained
from GO (Gene Ontology project), KEGG (Kyoto Encyclopedia
of Genes and Genomes) and mSigDB (Molecular Signature
Database), and the software DAVID (Database for Annotation
Visualization and Integrated discovery)38. Highlights of this
analysis revealed methylation differences induced by the FD diet
in promoter regions located in genes implicated in development
and with functions in the central nervous system, kidney, spleen,
digestive tract and muscle tissue (Table 2). In addition, the lowfolate diet also affected the methylation status of genes associated
with chronic diseases such as diabetes, autoimmune diseases,
neurological diseases, autism, schizophrenia and cancer (Table 3).
Less than 1% of the genome comprises imprinted genes that
are also subject to epigenetic reprogramming in spermatogenesis
and required for normal development of the embryo39. Imprinted
genes have been suggested to be sensitive to environmental
programming; however, none of the 65 imprinted genes tiled on
the promoter array displayed any signiﬁcant change in their DNA
methylation proﬁle.
Selected targets were analysed by Sequenom MassARRAY, a
technique based on bisulﬁte conversion with a resolution at
the CpG level. Criteria for the selection of gene targets for
validation were based on the following: ﬁrst, on variation in
methylation proﬁle to conﬁrm sensitivity over a range of
methylation states (low overall methylation, or intermediate
methylation or high overall methylation), which were detected
by the MeDIP-array as being differentially methylated at the
FDRo0.25; second, the gene had a biological link with
developmental processes that corresponded to developmental
abnormalities observed in offspring sired by FD males
(see Fig. 2); third, the gene had a suitable number of CpGs
for validation by the highly sensitive (single CpG resolution),
and quantitative method, Sequenom MassARRAY. The
genes selected for validation were the following: Rfwd2
(a ubiquitin-protein ligase expressed in the heart, testis,
stomach and muscle), Sﬁ1 (cell signalling, widespread tissue
distribution including the axial skeleton and muscle), Kdm3b
(a chromatin modiﬁer with widespread expression
during embryo development), Gm52 (required for placental
development) and Rbks (metabolism and cell signalling with
widespread tissue distribution; Fig. 4). Sequenom MassArray
analysis conﬁrmed signiﬁcantly altered DNA methylation at
speciﬁc CpG locations for selected genes, thereby validating
analysis by MeDIP-Array.
Histone modiﬁcations such as histone H3 lysine 4 (H3K4) and
lysine 9 (H3K9) methylation have been shown to differentially
mark genes in human and mouse sperm40,41,42. We investigated
whether folate availability could alter the sperm histone
modiﬁcation pattern by measuring global levels of methylation
of histone H3 at K9 and K4 in epididymal sperm extracts. Sperm
from FD males had signiﬁcantly reduced H3K4 and K9
monomethylation compared with FS sperm, and reduced H3K9
trimethylation compared with FS sperm (Po0.06 by Student’s
t-test; Supplementary Fig. S8). Thus, histone methylation levels in
sperm are sensitive to folate availability, and different histone
2
2
2
0
0
0
–2
2
–2
2
–2
2
0
0
0
–2
–2
Mir715
1 kb
Ddx58
–2
1 kb
FS
FD
Sfi1
0.5 kb
Figure 3 | Folate deﬁciency alters sperm DNA methylation. Changes in DNA methylation are illustrated by smoothed MeDIP over input log2 ratios of
individual oligonucleotides for the folate-sufﬁcient (FS) and the folate-deﬁcient (FD) animals. The gene is indicated at the bottom of the graph and the
arrow represents the transcription start site (TSS).
6
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Table 2 | Genes implicated in development were differentially methylated in sperm of folate-deﬁcient (FD) males compared with
the folate-sufﬁcient (FS) males.
Biological process
Central nervous system development
Behaviour
Ureteric bud development
Glomerulus development
Renal system process
Reproductive developmental process
Spleen development
Digestive tract morphogenesis
Regulation of muscle development
No. of genes differentially methylated
14
12
4
2
3
7
3
3
3
Selected genes
Arf4, Ets1, Foxp2, Hdac11, Helt, Lhx6, Nkx2-2
C3ar1, Fpr3, Helt, Hrh3, Htt, Nr4a3, Park7
Cd44, Slit2, Tcf21
Bcl2, Tcf21
Edn1, Uts2
Csf1, Srd5a1, Tcf21, Ube3a
Tcf21, Tlx1
Tcf21, Trp73
Edn1, Prox1
P-value
0.006
0.038
0.016
0.005
0.014
0.037
0.004
0.021
0.037
The MeDIP-ChIP assay was performed on sperm from FS males (n ¼ 3) and FD males (n ¼ 4 animals). The P-value was calculated by Fisher’s exact test.
Table 3 | Sperm from folate-deﬁcient males had differential methylation in comparison with folate-sufﬁcient males at sites
associated with cancer and chronic human diseases.
Diseases
Cancers (breast, colorectal, pancreatic, kidney,
leukaemia)
Diabetes type I
Diabetes type II
Autoimmune diseases (multiple sclerosis, lupus,
arthritis)
Neurological disorders (neural tube defects, Alzheimer’s
disease, Huntington’s disease)
Psychological disorders (autism, schizophrenia)
No. of genes
associated
18
3
3
6
10
7
methyltransferases may possess different sensitivities to an altered
methyl donor pool.
Altered gene expression in placenta of FD-sired offspring. To
determine whether there was transmission of epigenetic effects
from sires to offspring, global gene expression levels were assessed
in placenta from 18.5 dpc fetuses sired by either a FS or FD male
(Fig. 5). Analysis of array data revealed differential expression of
380 genes placenta from FD-sired offspring versus FS-sired offspring. The top 39 gene candidates selected based on their P-value
are shown in Fig. 5a. Functional analysis identiﬁed 21 of those
genes as implicated in the regulation of gene transduction/cell
signalling. Of these differentially expressed genes, 10 were selected for validation by real-time PCR in independent samples
(FS ¼ 8 placentas, FD ¼ 8 placentas each from different litters;
Fig. 5b,c). Notably, two validated genes with differential expression (Cav1, a cell cycle regulator, and Txndc16, a gene highly
expressed in placenta with a function in cell homoeostasis) were
also differentially methylated in sperm from FD males
(Supplementary Table S4). To determine whether the differential
DNA methylation observed in FD sperm at regions near Cav1
and Txndc16 were conserved in placenta, methylation levels were
measured by pyrosequencing after bisulﬁte conversion (Fig. 6).
No differences were observed between the FS- and FD-sired
fetuses’ placentas at those regions.
Discussion
In this study, we identify multiple regions of the sperm
epigenome that are environmentally programmed by factors in
the diet. There are three major epigenomic reprogramming
periods in the lifetime of male mammals. This massive resetting is
Genes
Abcg2, Bcl2, Tlx1, Ugt1a1, Ugt1a9, Arpc1a, Aff3, Cdc73,Cenpe, Ehmt1, Gstt2,
Folh1, Mtus1, Nme1, Slit2, Tyms, Whsc1l1, Nr4a3
Aff3, Tnfsf8, Ets1
Nkx2-2, Uts2, Cyp2e1
Bcl2, Cd44, Ebf1, Oaz1, Ets1, Gypa
Cep290, Cckar, Clock, Csf1, Cyp2e1, Htt, Lrrk2, Zdhhc15, Ube3a, Tyms
Cckar, Clock, Cyp2e1, Foxp2, Htt, Pnoc, Ube3a
essential to allow for the germ cell-speciﬁc epigenetic programme
to be established, which is required for embryo totipotency and
for the removal of epigenetic mutations to prevent the
transmission of disease. In the preimplantation embryo, the
epigenome is reprogrammed when DNA methylation and histone
methylation are actively removed with the exception of imprinted
genes, repeat sequences and unidentiﬁed regions that are involved
in the epigenetic inheritance of disease43,44. Epigenetic marks are
then re-acquired in a sex-, cell- and tissue-speciﬁc manner in the
peri-implantation period. The second period of reprogramming
also takes place in utero and the target is the primordial germ cells
within the developing embryo. As two major periods of
epigenomic programming occur in utero, this emphasizes that
this is a critical window in life for the induction of epigenetic
defects. The results of this study highlight the importance of the
in utero environment and show that dietary exposures in utero
affected the health of two generations: the fathers’ reproductive
health (F1) and the offspring (F2). The third period of
epigenomic reprogramming coincides with the onset of
spermatogenesis and spans from puberty to adulthood. It is
estimated that 1,000 spermatozoa are produced with every heart
beat45, and beginning with the stem cell suppliers up to the
mature sperm there are ongoing epigenomic programmes that
could be inﬂuenced by the environment. It remains to be
determined how sensitive epigenome reprogramming is in
spermatogenesis in the adults. The results of this study lend
further support to the notion that epigenetic marks in
spermatogenesis are dynamic and ﬂexible; thus, they can be
modiﬁed by nutritional, hormonal and toxin exposures46.
In this study, we demonstrate that a father’s FD diet altered the
sperm epigenome and that these changes were associated to genes
implicated in development and chronic disease. The functional
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889 | www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
7

9.
ARTICLE
Pctp
1.5
Fancg
Tmbim6
1
Cspp1
0.5
Tmem145
Dram1
0
Trib1
–0.5
Parp1
Plat
–1
Ddhd2
–1.5
Gm16894
Irgm2
Secisbp2
St13
Tpra1
6330578E17Rik
Fkrp
Zdhhc6
Mdn1
Prune
Txndc16
Slc2a4
Clec10a
Cav1
Ppfia3
Cd19
Col6a3
6430548M08Rik
Bicc1
Cav2
Olfr560
Cxcr1
Lrfn3
Fut7
Pirt
Plcl1
Faim3
Mtap1a
Ighv14–2
160
140
*
*
120
*
100
*
80
*
*
*
60
*
40
20
0
Clec10a Bicc1
mRNA levels/β-actin (% of control)
Folate
Deficient Sufficient
mRNA levels/β-actin (% of control)
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3889
Pirt Col6a3 Zbtb16 Plat
140
Fkrp
Prune
*
*
120
100
80
*
*
Folate-sufficient sired
Folate-deficient sired
60
40
20
0
Cav1
Txndc16
Figure 5 | Differential gene expression in placenta of offspring sired by FD versus FS males. (a) Heat-map showing the expression levels of 39 genes in
four placentas of 18.5 dpc fetuses sired by either an FS (n ¼ 4) or FD (n ¼ 4) male. Placentas analysed were from unique litters. (b) Validation of array
results by real-time PCR on an extended group of samples (n ¼ 8, FS and n ¼ 8, FD). (c) Selected array targets Cav1 and Txndc16 showed altered gene
expression and were differentially methylated in sperm of FD sires. Data are expressed as a percentage of the control b-actin, with the value of the FS at
100%. Means±s.e.m. of eight determinations are shown. For b and c, *Po0.05, **Po0.01 by Student’s t-test.
of folate availability differentially marks sperm for DNA
methylation. Does this then predispose his offspring to
metabolic conditions? The increasing rates of diabetes in the
USA from 6 million in 1985 to more than 20 million in 2010
indicate that the role of the sperm epigenome in this phenomena
and other chronic disease warrants deeper investigation.
Birth defects are the leading cause of infant mortality and
developmental disabilities in the United States. Current rates of
birth defects in Western society are 3%; however, the causes of
43% of those remain unknown56. It is well established that
exposure in utero to different lifestyle factors such as diet,
drugs or alcohol can alter embryo development. For instance, the
role of folate deﬁciency of the pregnant mother in neural tube
defects has long been known57. However, no studies have
investigated the role of paternal folate status and birth defects
in offspring. Here we demonstrate that defects in offspring are
associated with folate deﬁciency in the father. Previous studies
have linked sperm DNA damage to increased pregnancy loss and
embryo malformations58,59. To determine whether increased
embryo loss in the FD-sired pregnancies could be attributable to
increased DNA damage, we assessed DNA breaks in
spermatogenesis and sperm. Although there was an increased
incidence of DSBs as assess by g-H2AX foci, spermatocytes,
sperm was found to be free of DNA damage as assessed by Comet
assay. Thus, we suggest that the developmental abnormalities and
increased pregnancy loss in FD-sired pregnancies may be
attributable to the altered sperm epigenome. The wide range of
abnormalities observed in offspring correlated with DNA
methylation changes in sperm at a signiﬁcant number of genes
implicated in development. These included genes such as Lhx6
and Ets1, which function in the development of the central
nervous system, Tcf21 in kidney development and Prox1, which
regulates muscle development.
There is a paucity of information showing epigenetic
transmission through the germline. While our study indicates
that epigenetic transmission is a possibility, overlap between
genes that were identiﬁed as being differentially methylated in
sperm and differentially expressed in placenta was limited to two
genes. Moreover, these genes did not show methylation
differences in the placenta. The development of the placenta
and embryo is one of rapid cell division and differentiation. Thus,
the changes in the sperm that might be reﬂected in the embryonic
tissues may only be present for a particular window of
development making them difﬁcult to detect. Another possibility
is that mechanisms other than DNA methylation are involved
such as other chromatin modiﬁers. Notably, histone methylation
in sperm was also altered by folate deﬁciency, and histone
methylation has been localized to genes implicated in
development42,60.
These observations indicate that the male preconception diet
and overall health status may be of equal importance as the
mother’s and that the sperm epigenome plays a key role in the
development of the embryo as has been suggested by recent
studies41,42.
Importantly, this study indicates that there are environment
sensitive regions of the sperm epigenome that respond to diet and
transfer a so-called epigenomic map that inﬂuences development,
and perhaps in the long-term metabolism and disease in
offspring. This information opens new avenues of understanding
and preventing paternal routes to developmental defects and the
potential mechanisms underlying inter-generational disease
transmission.
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889 | www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
9

10.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3889
Antibodies. Primary antibodies used in this study were anti-H3K4-me1 (ab8895,
Abcam, 1:1000), anti-H3K9-me1 (ab8896, 1:1000), anti-H3K9me3 (07-442,
Millipore, 1:1000), anti-b-actin (A-1978, Sigma), anti-SYCP3 (ab15093; 1:100),
anti-gH2AX (05-636; 1:100), anti-MIS (sc-6886, Santa Cruz; 1:50), anti-mouse
VASA homologue (in house, 1:200), anti-3bHSD (sc-30820, 1:100) and anti-5methylcytidine (BI-MECY-0100, Eurogentec, 10 mg per immunoprecipitation
tube). Secondary antibodies were horseradish peroxidase (HRP)-conjugated donkey-anti-rabbit and anti-mouse antibodies (711-035-152 and 715-035-150, Jackson
Immunoresearch Laboratories, 1:500 for immunohistochemistry and 1:5000 for
western blotting), Alexa Fluor 488 goat-anti-mouse (A11001, Invitrogen, 1:1000)
and Alexa Fluor 594 goat-anti-rabbit (A11012, 1:1000).
30
Methylation level (%)
25
20
15
10
5
0
CpG
Txndc16
–4kb
CpG island
Region differentially
methylated in sperm
Folate-sufficient sired
Folate-deficient sired
80
Methylation level (%)
70
60
50
Homogenization-resistant sperm counts. The two caput epididymides were used
for each animal at 15 weeks (FS, n ¼ 5; FD, n ¼ 5). Tissues were thawed on ice in a
solution containing 0.5% Triton X-100 and then homogenized using a tissue
homogenizer. Finally, sperm heads were counted using a haemocytometer.
40
30
20
10
0
CpG
Cav1
CpG-rich region
+15kb
Region differentially
methylated in sperm
Figure 6 | Genomic regions located near Txndc16 and Cav1 that are
differentially methylated in FD sperm are not differentially methylated in
18.5 dpc placentas. Pyrosequencing analysis after bisulﬁte conversion was
carried out on 18.5 dpc placentas sired by FS and FD males (FS sired, n ¼ 4;
FD sired, n ¼ 4). The values are means ±s.e.m.
Methods
Animals and dietary treatments. To minimize genetic noise, the inbred C57BL/6
strain was used for generation of experimental males and for analysis of the sperm
epigenome. Breeding assessment of experimental males was done with the outbred
CD-1 strain (Charles River Laboratories, St-Constant, QC, Canada). Mice were
housed under a controlled light/dark cycle and were provided with food and water
ad libitum. All animal procedures were approved by the Animal Care and Use
Committee of McGill University, Montreal. Dietary exposures began in utero. To
generate experimental males, female C57BL/6 were fed either the FS (2 mg folic
acid per kg, n ¼ 69) diet (TD.01369, Harlan Laboratories, Madison, WI) or the FD
diet (0.3 mg folic acid per kg, n ¼ 64; TD.01546), 2 weeks before breeding with nonexperimental C57BL/6 males that were fed regular mouse chow (8640 Rodent diet;
used only for breeding to generate experimental males; Fig. 1). The effects of these
diets on serum levels of folate and homocysteine have been fully described in
different mouse strains21,61. To breed C57BL/6 females, C57BL/6 males were
brought to the females’ cages at night and removed in the morning to limit
consumption of the experimental diets by the males. Females were maintained on
the experimental diets through pregnancy and lactation. From weaning at PND21,
male pups were given the same experimental diet as their in utero exposure until
killing as adults.
10
Immunostaining. Tissues were ﬁxed in Bouin’s or 4% buffered formaldehyde
solution, processed for embedding in parafﬁn, and sectioned using standard histological protocols. Immunohistochemical staining was performed on 5-mm-thick
sections. Tissues were then deparafﬁnized and rehydrated. After washing in PBS
with 0.05% Brij for immunohistochemistry or PBS with 0.03% TritonX for
immunoﬂuorescence for 10 min, antigen retrieval was performed by incubating
tissue sections in sodium citrate buffer (pH 6.0), heating in the microwave until
boiling, followed by cooling for 30 min at room temperature. The slides were then
rinsed in PBS solution and endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature
only for immunohistochemistry. The sections were subsequently blocked in 5%
BSA in PBS solution for 1 h, and then incubated with the corresponding primary
antibody with rocking overnight at 4 °C. After washing, the sections were incubated
with secondary HRP-conjugated or ﬂuorescence-conjugated antibody for 1 h at
room temperature, followed by washing. For immunohistochemistry, immune
complexes were revealed by diaminobenzidine (Sigma) and sections were counterstained with haematoxylin. For immunoﬂuorescence, sections were mounted in
Vectashield (H1200, Vector) containing 40 ,6-diamidino-2-phenylindole. Reactivity
was viewed using a Nikon eclipse 80i microscope (Nikon, Mississauga, Canada).
Cell preparation for analysis of DNA DSBs. DNA DSBs were assessed in enriched pachytene spermatocyte cell spreads. Cells were prepared according to the
drying-down technique described by Peters et al.62. Tubules were removed from
testes and placed in hypotonic extraction buffer (30 mM Tris–HCl, 50 mM sucrose,
17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM dithiothreitol and
proteinase inhibitor cocktail; pH 8.2) for 60 min. Subsequently, tubules were
minced into a cloudy suspension in a sucrose solution (100 mM; pH 8.2), which
was then dispersed on slides pre-dipped in 1% paraformaldehyde. The slides were
washed in 0.4% Photoﬂo (Kodak) solution and dried. Co-immunoﬂuorescent
staining of SYCP3 and g-H2AX was then performed. Reactivity was visualized
using a confocal microscope Zeiss LSM 510–NLO.
Assessing DNA integrity in sperm using the COMET assay. DNA single- and
DSBs in spermatozoa of adult male mice were evaluated by the alkaline COMET
assay63. Brieﬂy, epididymal sperms were collected from 18-week-old FS and FD
mice (both n ¼ 3). Fifty microlitres of sperm suspension was mixed with 500 ml
0.7% molten low-melting point agarose at 42 °C. Fifty microlitres was immediately
pipetted and evenly spread onto slides. Cells were then lysed in lysis buffer (2.5 mol
NaCl, 100 mmol EDTA, 10 mmol Tris–HCl, 10% dimethyl sulfoxide, 1% Triton
X-100 and 40 mmol dithiothreitol; pH 10) for 1 h at 4 °C. After washing, slides were
incubated in an alkaline solution (1 mmol EDTA; pH 12.3) for 45 min at 4 °C.
Slides were then washed in 1 Â TAE buffer for 5 min before electrophoresis at
0.7 V cm À 1 for 10 min. Finally, the slides were dehydrated with 70% ethanol. DNA
was stained by 1:10,000 SYBR Green (Trevigen) and pictures were immediately
captured under microscope. For each animal, 100 cells were randomly selected and
measured using the KOMET 5.0 image analysis system (Kinetic Imaging Ltd,
Liverpool, UK).
Detection of apoptotic germ cells. Germ cell apoptosis was examined in testis
cross-sections from 15-week-old mice using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling detection protocol (Apoptag, Chemicon
International). All the tubules in each testis section were counted and the ratio of
tubules with a certain level of apoptotic cells (45, 410 or 415) to total number of
tubules counted were calculated.
Breeding trial. The effect of folate deﬁciency on male fertility was examined by
mating each experimental male (FS, n ¼ 20; FD, n ¼ 21; 8–10 weeks old) to a virgin
CD-1 female over a 5-day period. Females were examined daily for vaginal plugs.
Pregnancy rate was determined as the percentage of plug-positive females that
became pregnant.
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889 | www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.

11.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3889
Examination of fetuses and placentas. To determine whether the offspring
health was sensitive to paternal diet, CD-1 mice were mated to males fed either the
FS or FD diet throughout life (FS, n ¼ 32; FD, n ¼ 35; 2–3 months old) and
pregnancy outcomes were determined at 18.5 dpc. Females were killed and the
number of corpora lutea (CL) on the ovary was counted. The uteri were opened
and the number of resorption sites, implantation sites and live fetuses was
determined. Preimplantation loss was calculated as (CL À number of implants)/
CL Â 100 (ref. 64). Post-implantation loss was calculated as (number of
implants À viable fetuses)/number of implants Â 100.
Skeletal preparation and histopathology. Alcian blue and Alizarin red staining
of cleared skeletal preparations was performed according to Hogan et al.65. Fetuses
(18.5 dpc) were ﬁxed in 80% ethanol for 48 h. The skin and viscera were then
removed and the fetuses were further ﬁxed in 95% ethanol overnight and stained
with an alcian blue solution for 12 h. After washes in 95% ethanol for 5 h, the
fetuses were transferred in a 2% KOH solution for 24 h after which the muscles
were removed. The remaining bones and cartilages were stained in a 1% KOH with
0.015% alizarin red S solution for 12 h, washed for 2 days in a 1% KOH with 20%
glycerol solution and ﬁnally stored in glycerol. For histopathology, specimens were
ﬁxed in Bouin’s ﬂuid and embedded in parafﬁn. Deparafﬁnized sections were
stained with haematoxylin and eosin and analysed by McGill University
Comparative Pathology Services.
Methylation proﬁling by MeDIP-array. The MeDIP assay was performed as
described by Weber et al.66 on sperm from FS males (n ¼ 3) and FD males (n ¼ 4
animals). Genomic DNA was extracted with a DNeasy Mini Kit (Qiagen,
Mississauga, Canada). This DNA was then sonicated (Misonix) to obtain fragments
between 300 and 500 bp. Then, 4 mg of this sonicated genomic DNA was denatured
for 10 min at 95 °C and immunoprecipitated for 2 h at 4 °C with an anti
5-methylcytidine. Then, dynabeads coupled with a sheep anti-mouse-IgG antibody
(Dynal Biotech) for 2 h at 4 °C were added to the mixture. After washing,
proteinase K was added to the beads–DNA complex for 3 h at 50 °C and the
methylated DNA was isolated by phenolchloroform extraction followed by ethanol
precipitation. Input samples were processed in parallel. Immunoprecipitated DNA
and input DNA from MeDIP were ampliﬁed with the GenomePlex Complete
Whole Genome Ampliﬁcation Kit (Sigma, WGA2) according to the manufacturer’s
instructions. Array hybridization using the NimbleGen mouse 2.1 deluxe promoter
array was then carried out by Nimblegen.
Microarray analysis. Microarrays were processed and probe intensities were
extracted by Nimblegen. Extracted probe intensities were then analysed using the R
software environment for statistical computing. Log ratios of the bound (Cy5) and
input (Cy3) microarray channel intensities were computed for each microarray,
and then microarrays were normalized to one another using quantile normalization67 under the assumption that all samples have identical overall methylation
levels. The resulting values for each probe are called normalized intensities.
Differential methylation between groups of samples was determined in two stages
to ensure both statistical signiﬁcance and biological relevance. In the ﬁrst stage,
normalized probe intensity differences between FD and FS microarrays were
obtained for each probe (FD–FS). Given that the DNA samples were sonicated
before hybridization, we assumed that probes within 500 bp should approximately
agree. Genomic regions tiled with probes were partitioned into 1,000 bp regions,
each containing B10 probes. For each such 1,000 bp region, we calculated the
signiﬁcance of enrichment for high or low normalized probe differences of probes
within its promoter. Signiﬁcance was determined using the Wilcoxon rank-sum
test comparing differences of these probes against those of all the probes on the
microarray. The resulting P-values for each gene were then corrected for multiple
testing by calculating their FDR using the Benjamini and Hochberg algorithm. A
probe was then called differentially methylated if it satisﬁed each of the following:
the region containing it had a FDR of at most 0.1 and the normalized intensity
difference between FD and FS for the probe was greater than 1 or less than À 1.
The methylation level of a probe or site, when estimated from microarray data, was
obtained by applying a Bayesian deconvolution algorithm68 to normalized probe
intensities and corresponding sequence information indicating the locations of
CpG dinucleotides.
All functional analysis was based on gene sets obtained from GO, KEGG and
mSigDB. A gene was included in this analysis if at least one probe between
À 8,000 bp and 2,000 bp of its transcription start was called differentially
methylated. A small number of genes were initially included (Supplementary Table
S4); consequently, we relaxed the requirements for calling a probe differentially
methylated but increasing the FDR threshold from 0.1 to 0.25. Transcription start
sites for the mouse gene were obtained from BiomaRt (http://www.biomart.org/).
Functional signiﬁcance was determined by applying the hypergeometric to the
overlap between gene sets and genes called differentially methylated. Gene
expression levels from mouse testes were obtained from published data69. These
were used to partition genes by expression percentiles (0–5, 5–10,..., 95–100).
To obtain gene promoter methylation levels, we computed the average normalized
intensity of each probe across both microarrays and then applied the Bayesian
deconvolution algorithm mentioned above to the resulting averages.
Sequenom MassARRAY methylation analysis. One microgram of DNA from FS
(n ¼ 5) and FD (n ¼ 5) sperm was bisulﬁte treated with EZ DNA Methylation Gold
Kit (Zymoresearch, D5007). Primers to amplify different amplicons in speciﬁc
regions of Rfwd2 Sﬁ1 Kdm3b, Gm52 and Rbks were designed using the Sequenom
EpiDesigner application. Sequenom MassARRAY methylation analysis was then
performed using the MassARRAY Compact System (Sequenom, Inc. San Diego,
CA). This system is based on mass spectrometry analysis for qualitative and
quantitative detection of DNA methylation using homogeneous MassCLEAVE
base-speciﬁc cleavage and matrix-assisted laser desorption/ionization time-of-ﬂight
mass spectrometry. Spectra were elaborated by the Epityper software v1.2.0
(Sequenom, Inc.), which provides methylation values of each CpG unit expressed
as percentage. Those values result from the calculation of the ratio mass signals
between the methylated and non-methylated DNA.
Gene expression array. Total RNA was extracted from the placenta from different litters from 18.5 dpc fetuses sired by either a FS male (n ¼ 4) or a FD male
(n ¼ 4), using the RNeasy Mini Kit (Qiagen, Mississauga, Canada) according to the
manufacturer’s instructions. Cyanine 3-labelled CTP complementary RNA was
produced using the Low Input Quick Amp Labeling Kit, according to manufacturer’s instructions (Agilent Technologies, Mississauga, Canada). The labelled
complementary RNA was then normalized, fragmented and hybridized on
SurePrint G3 Mouse GE 8 Â 60 K arrays. The arrays were incubated in an Agilent
Hybridization oven at 65 °C for 17 h at 10 r.p.m. They were washed and scanned on
an Agilent DNA Microarray Scanner C. Output from the Agilent Feature
Extraction software were read into R, preprocessed and tested for differential
expression using functions from the Bioconductor package Limma. The normexp
method with an offset value of 16 was used for global background adjustment,
followed by quantile normalization and a log2 transformation. The annotation for
probes was retrieved from the Gene Expression Omnibus. Using the appropriate
Limma functions, a linear model was ﬁt to each gene separately. This linear model
included the paternal diet as a categorical variable. Moderated t-tests were performed on the difference between the FD and FS paternal diet groups. False discovery rate (FDR) estimation was carried out using the Benjamini–Hochberg
method. Candidates for validation were selected from a list of relaxed statistical
signiﬁcance, deﬁned as genes with Po0.01 (determined by the aforementioned
moderated t-tests) and a minimal absolute fold-change value of 1.2.
Reverse transcription and real-time PCR. Total RNA was extracted using the
RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and 1 mg of
total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Primers were designed
using Primer Express software (Applied Biosystems; Supplementary Table S2).
PCR reactions were performed following the SYBR Green Universal PCR Master
Mix protocol (Applied Biosystems) using an ABI Prism 7500 apparatus (Applied
Biosystems). Each sample was run in triplicate and negative controls (non-template
control and minus reverse transcription samples) were run for each primer pair.
The measured amount of each complementary DNA was normalized using the
control b-actin, a housekeeping gene with expression that was not altered by
treatment. The relative quantiﬁcation was performed using the standard curve
method. A list of the primers used is provided in Supplementary Table S5.
Western blotting. Western blots were performed to measure global levels of
histone methylation in FS and FD sperm. Sperm extracts were prepared in
Laemmli buffer (50 mM Tris–HCl (pH 7.5), 170 mM NaCl, 1% NP-40, 50 mM
NAF, 1 mM phenylmethylsulfonyl ﬂuoride, 100 mM NaVO3 and a proteinase
inhibitor cocktail). Equal amounts of protein were resolved by standard SDS–
polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were incubated overnight at 4 °C in PBS that contained 5%
low-fat milk, 0.05% Tween-20 and the corresponding ﬁrst antibody. After washing,
the membranes were incubated with a donkey-anti-rabbit or anti-mouse HRPconjugated secondary antibody diluted in 5% milk in PBS with 0.05% Tween-20,
and labelling was detected using enhanced chemiluminescence (Pierce). Membranes were exposed to Kodak autoradiography BioMax ﬁlm. Films were scanned
and the band intensity was quantiﬁed using AlphaDigiDocTM. Each experiment
was performed on six FD and six FS sperm samples and replicated a minimum of
three times. Full-size blots are shown in Supplementary Fig. S9.
Pyrosequencing. DNA methylation levels in placenta were determined by pyrosequencing using a PyroMark Q24 (Qiagen) after bisulﬁte conversion, which is a
real-time sequencing-based DNA analysis that quantiﬁes multiple and consecutive
CpG sites individually. Placental DNA was extracted using the DNeasy Mini Kit
(Qiagen); 1 mg of DNA was then bisulﬁte-treated using the EpiTect Fast DNA
Bisulﬁte Kit (Qiagen). Bisulﬁte-treated DNA was eluted in 15 ml volume, and 3 ml
was used for each PCR. PCR was performed using the HotStar DNA Polymerase
(Qiagen). The PCR was performed with one of the PCR primers biotinylated to
convert the PCR product to single-stranded DNA templates. The PCR products
(each 20 ml) were sequenced following the manufacturer’s instructions (Qiagen).
The methylation status of each locus was analysed individually as a T/C single-
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889 | www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
11